1. Field of the Invention
This invention relates to a technique of enrichment of decomposing bacteria which can be used to treat soil polluted by organic contaminants, such as agricultural chemicals, particularly to prevent groundwater pollution caused by agricultural chemicals in soil and a technique of isolating the decomposing bacteria by utilizing the enriching technique, and more particularly to techniques of these kinds which make it possible to drastically reduce time required for the enrichment or isolation of the decomposing bacteria.
2. Prior Art
To maintain today""s agricultural production, agricultural chemicals cannot be dispensed with, and to conserve flora in golf courses or the like as well, agricultural chemicals are used in large quantities. On the other hand, there is a concern that agricultural chemicals work as contaminants to have undesirable effects on the environment, especially to be a pollution source of groundwater. To eliminate this threat, it is desired to develop a technique of effectively preventing agricultural chemicals from remaining or spreading in the environment as contaminants after they have served their functions.
A great variety of microorganisms on the order of billions/1 g live in soils, and among these microorganisms there are not a few decomposing bacteria which are capable of decomposing organic compounds serving as functional skeletons in a lot of agricultural chemicals, thereby defusing the organic compounds or eliminating the same from the environment. Therefore, it is possible to thereby eliminate contaminants, such as agricultural chemicals or the like, from the environment by exploiting such capabilities of the decomposing bacteria. Generally, however, under natural conditions, decomposing bacteria capable of decomposing or degrading specific kinds of organic compounds are too thinly populated to effectively prevent contaminants from remaining or spreading in the environment. Therefore, a method of selectively enriching such decomposing bacteria and isolating the same from soil and thereafter applying the same to soil again is considered to be powerful means for preventing groundwater pollution by soil contaminated by organic compounds, such as agricultural chemicals and the like.
The method of selective enrichment/isolation of specific kinds of bacteria from diverse soil-inhabiting bacteria includes a soil percolation technique in which a column or the like is filled with soil containing inhabiting decomposing bacteria to form an enrichment soil layer, and inorganic salt medium, which contains only contaminants, such as agricultural chemicals, as solo carbon and nitrogen sources, is continuously circulated through the enrichment soil layer, whereby a specific kind of decomposing bacteria, that is, decomposing bacteria which are capable of using the carbon or nitrogen source contained in the inorganic salt medium for assimilation are selectively enriched for isolation. In the case of the above conventional soil percolation technique, generally a time period of one half to one year is required to enrich and isolate decomposing bacteria for practical use, which is a large impediment encountered in putting to practical use a groundwater pollution control technique and a soil cleanup technique using decomposing bacteria.
The present invention has been made under these circumstances, and its object is to provide a method of enriching decomposing bacteria which is capable of realizing bacteria-enriching/isolating speeds required high enough to put to practical use the groundwater pollution control technique using decomposing bacteria, and a method of isolating the decomposing bacteria by using the enriching method.
This invention is based on the following new finding: It is possible to improve the above soil percolation technique by adding an artificial microhabitat having a specific characteristic to an enrichment soil layer and thereby largely enhance the speed of enrichment of decomposing bacteria and the speed of isolation of the same from the soil.
According to the study of the present inventors, it has been found that if a porous material having an infinite number of micropores, such as charcoal, is fragmented to pieces of approximately several mm to ten and several mm in size such that the same can be handled with ease and at the same time has a large effective surface area, and then the fragmented porous material is mixed into an enrichment soil layer as an artificial microhabitat, decomposing bacteria can be effectively enriched and isolated over a time period of three weeks to three months, although they are slightly different depending on the kind of a contaminant and the kind of bacteria decomposing the contaminant. Such high-speed enrichment and isolation are not only far more excellent than the conventional methods requiring a time period of one half to one year, but also can sufficiently meet requirements for putting to use the technique of controlling pollution of groundwater by using the, decomposing bacteria.
Such high-speed enrichment and isolation is considered to be enabled by the following mechanism: First, the artificial microhabitat formed of the porous material efficiently adsorbs contaminants, that is, carbon and nitrogen sources, contained in an inorganic salt medium, into its infinite number of micropores mainly by hydrophobic adsorption to decompose bacteria. Further, under preferred conditions provided by the micropores of the artificial microhabitat, the decomposing bacteria can actively proliferate and be active in the micropores. These factors cooperate so that the decomposing bacteria in the micropores efficiently decompose or degrade the contaminants efficiently adsorbed in the micropores. As a result, the high-speed enrichment can be realized. In other words, by using the artificial microhabitat, the contaminants per se can be collected with efficiency in the artificial microhabitat, and moreover the artificial microhabitat can promote the growth of the decomposing bacteria, thereby enabling the high-speed enrichment of the bacteria. This also works effectively when artificial microhabitats containing enriched decomposing bacteria are used in farms and golf courses as a material for prevention of groundwater pollution, realizing a more efficient groundwater control capability.
In isolating decomposing bacteria through enhanced enrichment of the same, it goes without saying that the above-described mechanism works, but further, high-speed isolation of the decomposing bacteria is realized by isolating operations effectively carried out through the fact that enriched decomposing bacteria can be easily taken out from soil in the form of a treatable artificial microhabitat, and that by using the microhabitat, it is possible to easily inoculate the taken-out bacteria in new artificial microhabitats, and further that by circulation, it is possible to purify and enrich the bacteria in the artificial microhabitat.
One of the conditions for effectively activating the above-described mechanism is that a porous material as artificial microhabitats has a greater adsorptivity for adsorbing organic contaminants than a target soil.
According to the study of the present inventors, a more preferable condition of the porous material is that it has an adsorption coefficient (Kf) larger than scores of times and at the same time smaller than thousands of times as large as that of a soil used for an enrichment soil layer. Such a condition is substantially equivalent to a condition that the porous material has a specific surface area larger than approximately 50 m2/g and at the same time smaller than about 600 m2/g.
The reason for making these conditions preferable is related to the above-described mechanism. That is, if a porous material has an adsorptivity larger than required, it is impossible to cause adsorption of organic contaminants such that the decomposing bacteria are capable of effectively using the contaminants, more specifically, it is impossible to cause the organic contaminants to be efficiently adsorbed in micropores having suitable sizes for the bacteria to readily inhabit, in a state permitting the decomposing bacteria to make use of the organic contaminants. In other words, in such a case, an excessively large amount of organic contaminants are adsorbed in micropores other than ones where the bacteria can inhabit. Further, the excessively large adsorptivity of a porous material leads to degraded contaminant-decomposing efficiency thereof when the porous material containing the enriched decomposing bacteria is used as a material for prevention of groundwater pollution. That is, the requirement of an appropriate adsorptivity of a porous material contributes to an enhanced functionality of the porous material containing the enriched decomposing bacteria when it is used as a material for prevention of groundwater pollution, as well.
In addition to the above conditions, a predetermined condition set to distribution of the micropores in a porous material can make the above mechanism function more effectively. According to the study of the present inventors, this condition is that a volume ratio of micropores having sizes permitting the decomposing bacteria to form colonies to a total of micropores is equal to or larger than 10%. More specifically, when charcoal is taken as an example of the porous material, micropores thereof generally have a large range of diameter distribution from an order of nanometer to an order of hundreds of micrometer. of these micropores, those where decomposing bacteria are ready to settle and further capable of forming stable colonies have a diameter of 2 to 50 xcexcm, in general, and particularly, micropores having a diameter of 5 to 20 xcexcm are most suitable for decomposing bacteria. Therefore, a porous material having micropores of the above-mentioned sizes at a volume ratio equal to or higher than a predetermined value is suitable for an artificial microhabitat, and as the ratio becomes higher, the porous material becomes more suitable for an artificial microhabitat. Although this condition is somewhat related to the above-mentioned adsorptivity or specific surface area, it is not equivalent to any of these properties.
Therefore, the method of enriching decomposing bacteria according to the present invention aims to enrich a specific kind of bacteria capable of decomposing organic contaminants contained in soil. Hence, the enrichment method is characterized by comprising the steps of: mixing a fragmented porous material having an infinite number of micropores and a greater adsorptivity for adsorbing the organic contaminants than a target soil into a soil which the decomposing bacteria inhabit to form an enrichment soil layer, and circulating through the enrichment soil layer an inorganic salt medium containing carbon and nitrogen sources, the carbon and nitrogen sources being formed by only an organic contaminant to be decomposed, thereby enriching the decomposing bacteria in the fragmented porous material.
Further, in the enriching method according to the present invention, it is required as a preferred condition that the porous material has an adsorption coefficient (Kf) larger than scores of times and at the same time smaller than thousands of times as large as an adsorption coefficient (Kf) of the soil used for the enrichment soil layer, or that the porous material has a specific surface area larger than approximately 50 m2/g and at the same time smaller than 600 m2/g.
Further, in the enriching method according to the present invention, it is required as a more preferred condition that a volume ratio of micropores of the porous material having sizes permitting the decomposing bacteria to settle to a total of micropores of the porous material is equal to or larger than 10%.
The method of isolating decomposing bacteria according to the invention aims to isolate decomposing bacteria by using the enrichment method described above, and is characterized by comprising the steps of: inoculating a fragmented porous material in which the decomposing bacteria have already been enriched into a new fragmented porous material to form an enrichment layer consisting of the fragmented porous materials only, repeatedly carrying out a plurality of times an operation of enriching the decomposing bacteria in the new fragmented porous material as well by circulating through the enrichment layer an inorganic salt medium containing a carbon source and a nitrogen source, the carbon source and the nitrogen source being formed by only an organic contaminant to be. decomposed, thereby increasing a degree of purity and enrichment of the decomposing bacteria, for speedy isolation of the enriched decomposing bacteria.
As the porous material used in the present invention, there may be mentioned charcoal baked under a specific condition as a typical example. Especially, charcoal of broad-leaved tree, which is baked at a low or medium temperature of approximately 400 to 700xc2x0 C. is suitable and such charcoal satisfies the conditions concerning the adsorptivity and specific surface area as well as the micropore distribution, described above. Further, it is also possible to use a synthetic appetite material which has been made porous, and an inorganic foam, such as a ceramic foam. These porous materials are excellent in that none of them adversely affect soil.
The present invention is by no means limited to the above description, but the objects, advantages, features, and use of the invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. Further, it should be understood that all modifications properly made to the invention without departing from the spirit of the invention fall within the scope of the invention.